The worldwide demand for high potency sweeteners is increasing and, with blending of different sweeteners becoming a standard practice, the demand for alternatives is expected to increase. The sweet herb of Paraguay, Stevia rebaudiana Bertoni, produces an alternative sweetener with the added advantage that Stevia sweeteners are natural plant products. In addition, the sweet steviol glycosides have functional and sensory properties superior to those of many high potency sweeteners.
The sweet diterpene glycosides of Stevia have been characterized and eight sweet glycosides of steviol have been identified. These glycosides accumulate in Stevia leaves where they may comprise from 10 to 20% of the leaves dry weight. On a dry weight basis, a typical profile for the four major glycosides found in the leaves of Stevia comprises 0.3% dulcoside, 0.6% rebaudioside C, 3.8% rebaudioside A and 9.1% stevioside. Other glycosides identified within Stevia include rebaudioside B, C, and E, and dulcosides A and B. Rebaudioside B may be an artifact formed from rebaudioside A during extraction since both rebaudioside A and rebaudioside D are found to convert to rebaudioside B by alkaline hydrolysis.
Of the four major diterpene glycoside sweeteners present in Stevia leaves only two, stevioside and rebaudioside A, have had their physical and sensory properties well characterized. Stevioside and rebaudioside A were tested for stability in carbonated beverages and found to be both heat and pH stable (Chang and Cook, 1983). Stevioside is between 110 and 270 times sweeter than sucrose, rebaudioside A between 150 and 320 times sweeter than sucrose, and rebaudioside C between 40 and 60 times sweeter than sucrose. Dulcoside A was 30 times sweeter than sucrose. Rebaudioside A was the least astringent, the least bitter, had the least persistent aftertaste and was judged to have the most favorable sensory attributes of the four major steviol glycosides (Phillips, 1989 and Tanaka, 1997). Dubois and Stephanson (1984) have also confirmed that rebaudioside A is less bitter than stevioside and demonstrated that the bitter notes in stevioside and rebaudioside A are an inherent property of the compounds and not necessarily the result of impurities in whole plant extracts. Bitterness tends to increase with concentration for both stevioside and rebaudioside A. Both stevioside and rebaudioside A are synergistic in mixtures with other high potency sweeteners such as aspartame and are good candidates for inclusion in blends (Schiffman et al. 1995).
A process for the recovery of diterpene glycosides, including stevioside from the Stevia rebaudiana plant is described (U.S. Pat. No. 4,361,697). A variety of solvents, having different polarities, were used in a sequential treatment that concluded with a high performance liquid chromatographic (HPLC) separation procedure.
The method for the recovery of rebaudside A from the leaves of Stevia rebaudiana plants is developed (U.S. Pat. No. 4,082,858). Again, final purification is achieved by liquid chromatography subsequent followed by an initial extraction with water an alkanol having from 1 to 3 carbon carbons, preferably methanol. It is also disclosed that water may be used as the initial solvent, their preferred solvent at this stage is a liquid haloalkane having from 1 to 4 carbon atoms. The preferred second solvent is an alkanol having from 1 to 3 carbon atoms, while the preferred third solvent is an alkanol having from 1 to 4 carbon atoms and optionally minor amounts of water.
U.S. Pat. No. 4,892,938, to Giovanetto discloses a purification process in which the aqueous extracts of the plant are purified by passing these aqueous extracts through a series of ion-exchange resins which are selected to remove various impurities. The sweet glycosides remain in the water and are recovered by evaporation of the water. The advantage is that everything is done in water, while most other processes involve the use of a solvent at some point. The disadvantage is that the final product is quite impure with only about 70% is a mixture of the sweet glycosides. The balance is mainly material more polar than the sweet glycosides which we have identified as a complex mixture of polysaccharides (about 25%), and a small amount of yellow, oily material less polar than the sweet glycosides (about 5%).
The low polarity oil was isolated by chromatography. The flavor of the low polarity oil is very unpleasant. We have found this oil to be present in varying levels from 0.2 to 2.0% in every commercial product we have examined. Since of varying amounts this intensely off-flavored material is contained in the commercial materials it presents problems with quality control and flavor issues. The polysaccharide fraction also appears to contain off-flavor materials, but not as intense in flavor as the low polarity yellow oil.
The sweet glycosides obtained from Giovanetto process are always a mixture. We have determined that the two principle sweet glycosides are Stevioside and Rebaudioside A, and two of the minor sweet glycosides are Dulcoside and Rebaudioside C, although there are many other minor ones. We have isolated the two principle glycosides and we have found that there is a considerably different flavor between them with one being much more desirable than the other. Stevioside has an aftertaste which is undesirable. This aftertaste is present in Stevioside samples of even greater than 99% purity. On the other hand, Rebaudioside A does not possess an aftertaste and has a sweetness flavor comparable to sucrose. Thus it is recognized as having the most desirable sensory properties. In addition to this complexity, various impurities are also present and some of these possess undesirable flavors. The entire matter is further clouded by the extreme difficulty of doing analyzes. The analytical exercise pushes at the envelope of present technology and involves considerable art. Finally, the problem with the methods described above is that the resulting materials contain a mixture of all of the sweet glycosides.
The combined use of microfiltration, ultrafiltration, and nanofiltration is also applied for the purification of stevia extract (U.S. Pat. No. 5,972,120). The method gives a good result, however the application of the above mentioned equipments makes the product cost very expensive. Besides, the process again provided to isolate only the mixture of glycosides, but not pure individual compounds, such as stevioside and rebaudioside A.
Individual sweet glycosides are obtained from the stevia rebaudiana plant. A mixture of sweet glycosides extracted from the stevia rebaudiana plant is processed to remove impurities by using two ion exchange columns. After removing the mixed sweet glycosides from the second column with methanol the solution is dried. Upon refluxing the dried solids in a methanol solution and then cooling the solution, Stevioside precipitates out. The filtrate is further concentrated and cooled to precipitate out Rebaudioside A. This Rebaudioside A can be further purified as can the previously obtained Stevioside (U.S. Pat. No. 5,962,678). However, a large amount of toxic organic solvent, such as methanol is used.
However, stevioside possesses residual bitterness and aftertaste, which affect its qualitative characteristics. They can be eliminated by the reaction of intermolecular transglycosylation of various enzymes, upon which the attachment of new carbohydrates at positions C13 and C19 takes place. It is the number of carbohydrate units in the above-mentioned positions that determines the quality and degree of component's sweetness.
Pullulanase, isomaltase (Lobov et al., 1991), β-galactosidase (Kitahate et al., 1989), and dextrine saccharase (Yamamoto et al., 1994) are used as transglycosylating enzymes, with pullulan, maltose, lactose, and partially hydrolyzed starch, respectively, being as donors.
The treatment with pullulanase results in production of 13-O-[β-maltotriosyl-(1,2)-β-D-glucosyl]-19-O-β-D-glucosyl-steviol; 13-O-[β-maltosyl-(1,2)-β-D-glucosyl]-19-O-β-D-glucosyl-steviol and 13-O-[β-sephorosyl-19-O-β-maltotriosyl-steviol. Although the yields of the transglycosylated products were rather low, the selectivity in terms of the yield of the desirable mono- and di-derivatives was higher than in the case of CGTase (Lobov et al., 1991).
In case of maltase, three transglycosylated products are also produced, namely 13-O-[β-sephorosyl-19-O-β-isomaltosyl-steviol; 13-O-[β-isomaltosyl-(1,2)-β-D-glucosyl]-19-O-β-D-glucosyl-steviol and 13-O-[β-nigerosyl-(1,2)-β-D-glucosyl]-19-O-β-D-glucosyl-steviol.
The transglucosylation of stevioside was also done by action of cyclodextrin glucanotransferases (CGTase) produced by Bacillus stearothermophilus FERM-P No 2222 (U.S. Pat. No. 4,219,571). However, the commercialized stevioside consisting of roughly equal amounts of purified stevioside and lactose was used as substrate, and the transferring reaction is not investigated on pure stevioside, pure rebaudioside A, and purified stevia mixture.
The object of the present invention is to provide an advantageous process for the extraction of sweet glycosides from Stevia rebaudiana Bertoni plant, and for the isolation of stevioside and rebaudioside A, and a second object of the present invention is to provide a novel transferring enzyme produced by Themoactinomyces vulgaris and Bacillus halophilus which catalyzes the transglucosylation of stevioside and rebaudioside A, as well as the mixture of the glycosides obtained after extraction.